Pathogenic bacteria use various virulence mechanisms to infect hosts, including humans. Comparative studies based on genome sequencing of multiple pathogenic strains have suggested that bacterial species possess a pan-genome that is much larger than the genome of any single bacterium. This research focuses on the interaction between Pseudomonas syringae and plants. P. syringae delivers virulence factors called type III effector proteins into host plant cells using an evolutionarily conserved type III secretion system. This genus of pathogenic bacteria provides an excellent set of genomic and genetic tools to analyze co-evolution of host and pathogen, from the level of genome organization and horizontal gene flow down to the level of the interaction between pathogen virulence proteins and their host targets. Animal pathogens that utilize the type III secretion system include Salmonella spp., Yersinia spp., Shigella spp. and pathogenic E. coli. Thus, the results from this research will inform evolutionary and mechanistic studies of these bacteria and their animal hosts, including humans. The first grant period was devoted to methods for rapidly identifying P. syringae type III effector genes and their products. The main goals were completed and additional biological studies focused on particular type III effector (virulence) factor families was facilitated. In this competitive renewal, it is proposed to continue to use functional genomics and high throughput next generation genome sequencing to dissect both the mechanisms and evolution of virulence in P. syringae. In addition, this proposal presents plausible evidence that comparative genomics and molecular evolutionary methods can be used to identify novel candidate virulence genes. Our ultimate goal is to identify the pan genome of P. syringae and characterize both type III effectors and novel virulence factors across this pan-genome. This goal will provide both methods and data that will inform similar studies of human bacterial pathogens that continue to kill and sicken millions each year around the world. Project Narrative: Pathogenic bacteria use various virulence mechanisms to infect hosts, including humans. Bacterial pathogens cause a variety of human diseases and kill millions of people each year around the world. Bacterial disease of plants is also important, and they lead to loss of crops with values of several billion dollars worldwide each year. Crop loss further therefore directly adds to threats to human health, particularly in developing countries where the disease pressure from both bacterial pathogens of humans and of plants converge. Loss of crops is also tied to human health because those lost crops used precious water before they were infected and rendered useless as food or fodder. Thus, crop loss impacts human health by impacting water use efficiency in human populations. Plant pathogens like the one that is the focus of this proposal and animal pathogens including Salmonella spp., Yersinia spp., Shigella spp. and pathogenic E. coli all use a similar mechanism to infect their hosts. Thus, the results from this research will inform evolutionary and mechanistic studies of these bacteria and their animal hosts, including humans.